Lettuce variety SV2867LG

ABSTRACT

The invention provides seed and plants of the lettuce line SV2867LG. The invention thus relates to the plants, seeds, and tissue cultures of lettuce line SV2867LG, and to methods for producing a lettuce plant produced by crossing a plant of lettuce line SV2867LG with itself or with another lettuce plant, such as a plant of another genotype. The invention further relates to seeds and plants produced by such crossing. The invention further relates to parts of a plant of lettuce line SV2867LG, including the gametes of such plants.

FIELD OF THE INVENTION

The present invention relates to the field of plant breeding and, morespecifically, to the development of lettuce line SV2867LG.

BACKGROUND OF THE INVENTION

The goal of vegetable breeding is to combine various desirable traits ina single variety/hybrid. Such desirable traits may include any traitdeemed beneficial by a grower and/or consumer including greater yield,resistance to insects or pathogens, tolerance to environmental stress,better agronomic quality, higher nutritional value, growth rate andfruit properties.

Breeding techniques take advantage of a plant's method of pollination.There are two general methods of pollination: a plant self-pollinates ifpollen from one flower is transferred to the same or another flower ofthe same plant or plant variety. A plant cross-pollinates if pollencomes to it from a flower of a different plant variety.

Plants that have been self-pollinated and selected for type over manygenerations become homozygous at almost all genetic loci and produce auniform population of true breeding progeny, a homozygous plant. A crossbetween two such homozygous plants of different genotypes produces auniform population of hybrid plants that are heterozygous for manygenetic loci. Conversely, a cross of two plants each heterozygous at anumber of loci produces a population of hybrid plants that differgenetically and are not uniform. The resulting non-uniformity makesperformance unpredictable.

The development of uniform varieties requires the development ofhomozygous inbred plants, the crossing of these inbred plants, and theevaluation of the crosses. Pedigree breeding and recurrent selection areexamples of breeding methods that have been used to develop inbredplants from breeding populations. Those breeding methods combine thegenetic backgrounds from two or more plants or various other broad-basedsources into breeding pools from which new lines are developed byselfing and selection of desired phenotypes. The new lines are evaluatedto determine which of those have commercial potential.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a lettuce plant of theline SV2867LG. Also provided are lettuce plants having all thephysiological and morphological characteristics of lettuce lineSV2867LG. Parts of the lettuce plant of the present invention are alsoprovided, for example, including pollen, an ovule, an embryo, a seed,and a cell of the plant.

The invention also concerns seed of lettuce line SV2867LG. The lettuceseed of the invention may be provided as an essentially homogeneouspopulation of lettuce seed of the line designated SV2867LG. Essentiallyhomogeneous populations of seed are generally free from substantialnumbers of other seed. Therefore, in one embodiment, seed of lineSV2867LG may be defined as forming at least about 97% of the total seed,including at least about 98%, 99%, or more of the seed. The populationof lettuce seed may be particularly defined as being essentially freefrom hybrid seed. The seed population may be separately grown to providean essentially homogeneous population of lettuce plants designatedSV2867LG.

In another aspect of the invention, a plant of lettuce line SV2867LGcomprising an added heritable trait is provided. The heritable trait maycomprise a genetic locus that is, for example, a dominant or recessiveallele. In one embodiment of the invention, a plant of lettuce lineSV2867LG is defined as comprising a single locus conversion. In specificembodiments of the invention, an added genetic locus confers one or moretraits such as, for example, herbicide tolerance, insect resistance,disease resistance, and modified carbohydrate metabolism. In furtherembodiments, the trait may be, for example, conferred by a naturallyoccurring gene introduced into the genome of the line by backcrossing, anatural or induced mutation, or a transgene introduced through genetictransformation techniques into the plant or a progenitor of any previousgeneration thereof. When introduced through transformation, a geneticlocus may comprise one or more genes integrated at a single chromosomallocation.

In some embodiments, a single locus conversion includes one or moresite-specific changes to the plant genome, such as, without limitation,one or more nucleotide modifications, deletions, or insertions. A singlelocus may comprise one or more genes or nucleotides integrated ormutated at a single chromosomal location. In one embodiment, a singlelocus conversion may be introduced by a genetic engineering technique,methods of which include, for example, genome editing with engineerednucleases (GEEN). Engineered nucleases include, but are not limited to,Cas endonucleases; zinc finger nucleases (ZFNs); transcriptionactivator-like effector nucleases (TALENs); engineered meganucleases,also known as homing endonucleases; and other endonucleases for DNA orRNA-guided genome editing that are well-known to the skilled artisan.

In another aspect of the invention, a tissue culture of regenerablecells of a plant of line SV2867LG is provided. The tissue culture willpreferably be capable of regenerating plants capable of expressing allof the physiological and morphological characteristics of the startingplant, and of regenerating plants having substantially the same genotypeas the starting plant. Examples of some of the physiological andmorphological characteristics of the line SV2867LG include those traitsset forth in the table herein. The regenerable cells in such tissuecultures may be derived, for example, from embryos, meristems,cotyledons, pollen, leaves, anthers, roots, root tips, pistil, flower,seed and stalks. Still further, the present invention provides lettuceplants regenerated from a tissue culture of the invention, the plantshaving all the physiological and morphological characteristics of lineSV2867LG.

In yet another aspect of the invention, processes are provided forproducing lettuce seeds and plants, which processes generally comprisecrossing a first parent lettuce plant with a second parent lettuceplant, wherein at least one of the first or second parent lettuce plantsis a plant of the line designated SV2867LG. These processes may befurther exemplified as processes for preparing hybrid lettuce seed orplants, wherein a first lettuce plant is crossed with a second lettuceplant of a different, distinct genotype to provide a hybrid that has, asone of its parents, the lettuce plant line SV2867LG. In these processes,crossing will result in the production of seed. The seed productionoccurs regardless of whether the seed is collected or not.

In one embodiment of the invention, the first step in “crossing”comprises planting seeds of a first and second parent lettuce plant,often in proximity so that pollination will occur for example, mediatedby insect vectors. Alternatively, pollen can be transferred manually.Where the plant is self-pollinated, pollination may occur without theneed for direct human intervention other than plant cultivation.

A second step may comprise cultivating or growing the seeds of first andsecond parent lettuce plants into plants that bear flowers. A third stepmay comprise preventing self-pollination of the plants, such as byemasculating the male portions of flowers, (i.e., treating ormanipulating the flowers to produce an emasculated parent lettuceplant). Self-incompatibility systems may also be used in some hybridcrops for the same purpose. Self-incompatible plants still shed viablepollen and can pollinate plants of other varieties but are incapable ofpollinating themselves or other plants of the same line.

A fourth step for a hybrid cross may comprise cross-pollination betweenthe first and second parent lettuce plants. Yet another step comprisesharvesting the seeds from at least one of the parent lettuce plants. Theharvested seed can be grown to produce a lettuce plant or hybrid lettuceplant.

The present invention also provides the lettuce seeds and plantsproduced by a process that comprises crossing a first parent lettuceplant with a second parent lettuce plant, wherein at least one of thefirst or second parent lettuce plants is a plant of the line designatedSV2867LG. In one embodiment of the invention, lettuce seed and plantsproduced by the process are first generation (F₁) hybrid lettuce seedand plants produced by crossing a plant in accordance with the inventionwith another, distinct plant. The present invention further contemplatesplant parts of such an F₁ hybrid lettuce plant, and methods of usethereof. Therefore, certain exemplary embodiments of the inventionprovide an F₁ hybrid lettuce plant and seed thereof.

In still yet another aspect of the invention, the genetic complement ofthe lettuce plant line designated SV2867LG is provided. The phrase“genetic complement” is used to refer to the aggregate of nucleotidesequences, the expression of which sequences defines the phenotype of,in the present case, a lettuce plant, or a cell or tissue of that plant.A genetic complement thus represents the genetic makeup of a cell,tissue or plant, and a hybrid genetic complement represents the geneticmake-up of a hybrid cell, tissue or plant. The invention thus provideslettuce plant cells that have a genetic complement in accordance withthe lettuce plant cells disclosed herein, and plants, seeds and plantscontaining such cells.

Plant genetic complements may be assessed by genetic marker profiles,and by the expression of phenotypic traits that are characteristic ofthe expression of the genetic complement, e.g., isozyme typing profiles.It is understood that line SV2867LG or a first generation progenythereof could be identified by any of the many well-known techniquessuch as, for example, Simple Sequence Length Polymorphisms (SSLPs)(Williams et al., Nucleic Acids Res., 1 8:6531-6535, 1990), RandomlyAmplified Polymorphic DNAs (RAPDs), DNA Amplification Fingerprinting(DAF), Sequence Characterized Amplified Regions (SCARs), ArbitraryPrimed Polymerase Chain Reaction (AP-PCR), Amplified Fragment LengthPolymorphisms (AFLPs) (EP 534 858, specifically incorporated herein byreference in its entirety), and Single Nucleotide Polymorphisms (SNPs)(Wang et al., Science, 280:1077-1082, 1998).

In still yet another aspect, the present invention provides hybridgenetic complements, as represented by lettuce plant cells, tissues,plants, and seeds, formed by the combination of a haploid geneticcomplement of a lettuce plant of the invention with a haploid geneticcomplement of a second lettuce plant, preferably, another, distinctlettuce plant. In another aspect, the present invention provides alettuce plant regenerated from a tissue culture that comprises a hybridgenetic complement of this invention.

In still yet another aspect, the invention provides a method ofdetermining the genotype of a plant of lettuce line SV2867LG comprisingdetecting in the genome of the plant at least a first polymorphism. Themethod may, in certain embodiments, comprise detecting a plurality ofpolymorphisms in the genome of the plant. The method may furthercomprise storing the results of the step of detecting the plurality ofpolymorphisms on a computer readable medium. The invention furtherprovides a computer readable medium produced by such a method.

In still yet another aspect, the present invention provides a method ofproducing a plant derived from line SV2867LG, the method comprising thesteps of: (a) preparing a progeny plant derived from line SV2867LG,wherein said preparing comprises crossing a plant of the line SV2867LGwith a second plant; and (b) crossing the progeny plant with itself or asecond plant to produce a seed of a progeny plant of a subsequentgeneration. In further embodiments, the method may additionallycomprise: (c) growing a progeny plant of a subsequent generation fromsaid seed of a progeny plant of a subsequent generation and crossing theprogeny plant of a subsequent generation with itself or a second plant;and repeating the steps for an additional 3-10 generations to produce aplant derived from line SV2867LG. The plant derived from line SV2867LGmay be an inbred line, and the aforementioned repeated crossing stepsmay be defined as comprising sufficient inbreeding to produce the inbredline. In the method, it may be desirable to select particular plantsresulting from step (c) for continued crossing according to steps (b)and (c). By selecting plants having one or more desirable traits, aplant derived from line SV2867LG is obtained which possesses some of thedesirable traits of the line as well as potentially other selectedtraits.

In certain embodiments, the present invention provides a method ofproducing food comprising: (a) obtaining a plant of lettuce lineSV2867LG, wherein the plant has been cultivated to maturity, and (b)collecting leaf tissue from the plant, wherein the leaf tissue iscapable of use as food.

Any embodiment discussed herein with respect to one aspect of theinvention applies to other aspects of the invention as well, unlessspecifically noted.

The term “about” is used to indicate that a value includes the standarddeviation of the mean for the device or method being employed todetermine the value. The use of the term “or” in the claims is used tomean “and/or” unless explicitly indicated to refer to alternatives onlyor the alternatives are mutually exclusive. When used in conjunctionwith the word “comprising” or other open language in the claims, thewords “a” and “an” denote “one or more,” unless specifically notedotherwise. The terms “comprise,” “have” and “include” are open-endedlinking verbs. Any forms or tenses of one or more of these verbs, suchas “comprises,” “comprising,” “has,” “having,” “includes” and“including,” are also open-ended. For example, any method that“comprises,” “has” or “includes” one or more steps is not limited topossessing only those one or more steps and also covers other unlistedsteps. Similarly, any plant that “comprises,” “has” or “includes” one ormore traits is not limited to possessing only those one or more traitsand covers other unlisted traits.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and any specificexamples provided, while indicating specific embodiments of theinvention, are given by way of illustration only, since various changesand modifications within the spirit and scope of the invention willbecome apparent to those skilled in the art from this detaileddescription.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods and compositions relating to plants,seeds and derivatives of the lettuce line designated SV2867LG. This lineshows uniformity and stability within the limits of environmentalinfluence for the traits described hereinafter. Lettuce line SV2867LGprovides sufficient seed yield. By crossing with a distinct secondplant, uniform F₁ hybrid progeny can be obtained.

Lettuce line SV2867LG, also known as 13-8W-CRC-0004, was developed fromthe initial cross of an iceberg lettuce variety and a romaine lettucevariety. Lettuce line SV2867LG has a crunchy texture, sweet taste, andunique shape with oblong leaves. Lettuce line SV2867LG also comprisesone or more of the romaine lettuce characteristics described inInternational Patent Publication No. WO/2002078428.

A. Physiological and Morphological Characteristics of Lettuce LineSV2867LG

In accordance with one aspect of the present invention, there isprovided a plant having the physiological and morphologicalcharacteristics of lettuce line SV2867LG. A description of thephysiological and morphological characteristics of lettuce line SV2867LGis presented in Table 1.

TABLE 1 Physiological and Morphological Characteristics of Lettuce LineSV2867LG CHARACTERISTIC SV2867LG SV3176LG SV6881LG Plant spread of frameleaves (cm) 33.6 33.1 33.6 diameter small small small fasciation (atflowering absent absent absent stage) head, degree of overlapping absentor absent or weak of upper part of plant very weak very weak headdiameter (cm) 13.0 11.7 11.7 number of leaves (varieties medium manymany with degree of overlapping of upper part of leaves: absent or weak)number of leaves (in whole 47   49   51   number) axillary sproutingweak weak absent Bolting time of beginning of bolting late very earlymedium under long day conditions Leaf leaf blade, density of dense densedense incisions on margin leaf blade, venation not flabellate notflabellate not flabellate intensity of green color medium medium medium(harvest mature outer leaves) anthocyanin coloration absent or absent orabsent or very weak very weak very weak color green green yellowishgreen glossiness of upper side strong strong strong blistering (harvestmature absent/slight absent/slight absent/slight outer leaves) or veryweak or very weak or very weak thickness (harvest mature thick thickthick outer leaves) attitude at harvest maturity erect erect erect(outer leaves from head lettuce or adult leaves from cutting and stemlettuce) shape medium medium medium elliptic elliptic elliptic shape ofapex obtuse obtuse rounded shape of fourth leaf elongated elongatedelongated undulation of margin medium medium medium type of incisions ofmargin regularly regularly regularly dentate dentate dentate depth ofincisions of margin shallow shallow shallow number of divisions (outerabsent or absent or absent or leaves from head lettuce or very few veryfew very few adult leaves from cutting and stem lettuce) longitudinalsection concave concave concave (number of divisions: absent or veryfew) butt, shape slightly slightly slightly concave concave concavebutt, midrib moderately prominently prominently raised raised raisedSeed color black black black Seedling shape of cotyledon intermediateintermediate intermediate These are typical values. Values may vary dueto environment. Other values that are substantially equivalent arewithin the scope of the invention.

B. Breeding Lettuce Plants

One aspect of the current invention concerns methods for crossing thelettuce line SV2867LG with itself or a second plant and the seeds andplants produced by such methods. These methods can be used forpropagation of line SV2867LG, or can be used to produce hybrid lettuceseeds and the plants grown therefrom. Hybrid seeds are produced bycrossing line SV2867LG with second lettuce parent line.

The development of new varieties using one or more starting varieties iswell known in the art. In accordance with the invention, novel varietiesmay be created by crossing line SV2867LG followed by multiplegenerations of breeding according to such well-known methods. Newvarieties may be created by crossing with any second plant. In selectingsuch a second plant to cross for the purpose of developing novel lines,it may be desired to choose those plants which either themselves exhibitone or more selected desirable characteristics or which exhibit thedesired characteristic(s) in progeny. Once initial crosses have beenmade, inbreeding and selection take place to produce new varieties. Fordevelopment of a uniform line, often five or more generations of selfingand selection are involved.

Uniform lines of new varieties may also be developed by way ofdouble-haploids. This technique allows the creation of true breedinglines without the need for multiple generations of selfing andselection. In this manner true breeding lines can be produced in aslittle as one generation. Haploid embryos may be produced frommicrospores, pollen, anther cultures, or ovary cultures. The haploidembryos may then be doubled autonomously, or by chemical treatments(e.g. colchicine treatment). Alternatively, haploid embryos may be growninto haploid plants and treated to induce chromosome doubling. In eithercase, fertile homozygous plants are obtained. In accordance with theinvention, any of such techniques may be used in connection with lineSV2867LG and progeny thereof to achieve a homozygous line.

New varieties may be created, for example, by crossing line SV2867LGwith any second plant and selection of progeny in various generationsand/or by doubled haploid technology. In choosing a second plant tocross for the purpose of developing novel lines, it may be desired tochoose those plants which either themselves exhibit one or more selecteddesirable characteristics or which exhibit the desired characteristic(s)in progeny. After one or more lines are crossed, true-breeding lines maybe developed.

Backcrossing can also be used to improve an inbred plant. Backcrossingtransfers a specific desirable trait from one inbred or non-inbredsource to an inbred that lacks that trait. This can be accomplished, forexample, by first crossing a superior inbred (A) (recurrent parent) to adonor inbred (non-recurrent parent), which carries the appropriate locusor loci for the trait in question. The progeny of this cross are thenmated back to the superior recurrent parent (A) followed by selection inthe resultant progeny for the desired trait to be transferred from thenon-recurrent parent. After five or more backcross generations withselection for the desired trait, the progeny have the characteristicbeing transferred, but are like the superior parent for most or almostall other loci. The last backcross generation would be selfed to givepure breeding progeny for the trait being transferred.

The line of the present invention is particularly well suited for thedevelopment of new lines based on the elite nature of the geneticbackground of the line. In selecting a second plant to cross withSV2867LG for the purpose of developing novel lettuce lines, it willtypically be preferred to choose those plants which either themselvesexhibit one or more selected desirable characteristics or which exhibitthe desired characteristic(s) when in hybrid combination. Examples ofdesirable characteristics may include, for example, seed yield, seedsize, seed shape, seed uniformity, early maturity, disease resistance,herbicide tolerance, seedling vigor, adaptability for soil conditions,and adaptability for climate conditions.

C. Further Embodiments of the Invention

In certain aspects of the invention, plants described herein modified toinclude at least a first desired heritable trait are provided. Suchplants may, in one embodiment, be developed by a plant breedingtechnique called backcrossing, wherein essentially all of the desiredmorphological and physiological characteristics of a variety arerecovered in addition to a genetic locus transferred into the plant viathe backcrossing technique. The term single locus converted plant asused herein refers to those lettuce plants which are developed by aplant breeding technique called backcrossing or by genetic engineering,wherein essentially all of the desired morphological and physiologicalcharacteristics of a variety are recovered or conserved in addition tothe single locus introduced into the variety via the backcrossing orgenetic engineering technique, respectively. By essentially all of themorphological and physiological characteristics, it is meant that thecharacteristics of a plant are recovered or conserved that are otherwisepresent when compared in the same environment, other than an occasionalvariant trait that might arise during backcrossing, introduction of atransgene, or application of genetic engineering. It is understood thata locus introduced by backcrossing may or may not be transgenic inorigin, and thus the term backcrossing specifically includesbackcrossing to introduce loci that were created by introduction of atransgene or application of genetic engineering.

Backcrossing methods can be used with the present invention to improveor introduce a characteristic into the present variety. The parentallettuce plant which contributes the locus for the desired characteristicis termed the nonrecurrent or donor parent. This terminology refers tothe fact that the nonrecurrent parent is used one time in the backcrossprotocol and therefore does not recur. The parental lettuce plant towhich the locus or loci from the nonrecurrent parent are transferred isknown as the recurrent parent as it is used for several rounds in thebackcrossing protocol.

In a typical backcross protocol, the original variety of interest(recurrent parent) is crossed to a second variety (nonrecurrent parent)that carries the single locus of interest to be transferred. Theresulting progeny from this cross are then crossed again to therecurrent parent and the process is repeated until a lettuce plant isobtained wherein essentially all of the desired morphological andphysiological characteristics of the recurrent parent are recovered inthe converted plant, in addition to the single transferred locus fromthe nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalvariety. To accomplish this, a single locus of the recurrent variety ismodified or substituted with the desired locus from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphologicalconstitution of the original variety. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross; one ofthe major purposes is to add some commercially desirable trait to theplant. The exact backcrossing protocol will depend on the characteristicor trait being altered and the genetic distance between the recurrentand nonrecurrent parents. Although backcrossing methods are simplifiedwhen the characteristic being transferred is a dominant allele, arecessive allele, or an additive allele (between recessive anddominant), may also be transferred. In this instance it may be necessaryto introduce a test of the progeny to determine if the desiredcharacteristic has been successfully transferred.

In one embodiment, progeny lettuce plants of a backcross in whichSV2867LG is the recurrent parent comprise (i) the desired trait from thenon-recurrent parent and (ii) all of the physiological and morphologicalcharacteristics of lettuce line SV2867LG as determined at the 5%significance level when grown in the same environmental conditions.

Lettuce varieties can also be developed from more than two parents. Thetechnique, known as modified backcrossing, uses different recurrentparents during the backcrossing. Modified backcrossing may be used toreplace the original recurrent parent with a variety having certain moredesirable characteristics or multiple parents may be used to obtaindifferent desirable characteristics from each.

With the development of molecular markers associated with particulartraits, it is possible to add additional traits into an established germline, such as represented here, with the end result being substantiallythe same base germplasm with the addition of a new trait or traits.Molecular breeding, as described in Moose and Mumm, 2008 (PlantPhysiol., 147: 969-977), for example, and elsewhere, provides amechanism for integrating single or multiple traits or QTL into an eliteline. This molecular breeding-facilitated movement of a trait or traitsinto an elite line may encompass incorporation of a particular genomicfragment associated with a particular trait of interest into the eliteline by the mechanism of identification of the integrated genomicfragment with the use of flanking or associated marker assays. In theembodiment represented here, one, two, three or four genomic loci, forexample, may be integrated into an elite line via this methodology. Whenthis elite line containing the additional loci is further crossed withanother parental elite line to produce hybrid offspring, it is possibleto then incorporate at least eight separate additional loci into thehybrid. These additional loci may confer, for example, such traits as adisease resistance or a fruit quality trait. In one embodiment, eachlocus may confer a separate trait. In another embodiment, loci may needto be homozygous and exist in each parent line to confer a trait in thehybrid. In yet another embodiment, multiple loci may be combined toconfer a single robust phenotype of a desired trait.

Many single locus traits have been identified that are not regularlyselected for in the development of a new inbred but that can be improvedby backcrossing techniques. Single locus traits may or may not betransgenic; examples of these traits include, but are not limited to,male sterility, herbicide resistance, resistance to bacterial, fungal,or viral disease, insect resistance, restoration of male fertility,modified fatty acid or carbohydrate metabolism, and enhanced nutritionalquality. These comprise genes generally inherited through the nucleus.

Direct selection may be applied where the single locus acts as adominant trait. An example of a dominant trait is the downy mildewresistance trait. For this selection process, the progeny of the initialcross are sprayed with downy mildew spores prior to the backcrossing.The spraying eliminates any plants which do not have the desired downymildew resistance characteristic, and only those plants which have thedowny mildew resistance gene are used in the subsequent backcross. Thisprocess is then repeated for all additional backcross generations.

Selection of lettuce plants for breeding is not necessarily dependent onthe phenotype of a plant and instead can be based on geneticinvestigations. For example, one can utilize a suitable genetic markerwhich is closely genetically linked to a trait of interest. One of thesemarkers can be used to identify the presence or absence of a trait inthe offspring of a particular cross, and can be used in selection ofprogeny for continued breeding. This technique is commonly referred toas marker assisted selection. Any other type of genetic marker or otherassay which is able to identify the relative presence or absence of atrait of interest in a plant can also be useful for breeding purposes.Procedures for marker assisted selection applicable to the breeding oflettuce are well known in the art. Such methods will be of particularutility in the case of recessive traits and variable phenotypes, orwhere conventional assays may be more expensive, time consuming orotherwise disadvantageous. In addition, marker assisted selection may beused to identify plants comprising desirable genotypes at the seed,seedling, or plant stage, to identify or assess the purity of acultivar, to catalog the genetic diversity of a germplasm collection,and to monitor specific alleles or haplotypes within an establishedcultivar.

Types of genetic markers which could be used in accordance with theinvention include, but are not necessarily limited to, Simple SequenceLength Polymorphisms (SSLPs) (Williams et al., Nucleic Acids Res., 18:6531-6535, 1990), Randomly Amplified Polymorphic DNAs (RAPDs), DNAAmplification Fingerprinting (DAF), Sequence Characterized AmplifiedRegions (SCARs), Arbitrary Primed Polymerase Chain Reaction (AP-PCR),Amplified Fragment Length Polymorphisms (AFLPs) (EP 534 858,specifically incorporated herein by reference in its entirety), andSingle Nucleotide Polymorphisms (SNPs) (Wang et al., Science,280:1077-1082, 1998).

In particular embodiments of the invention, marker assisted selection isused to increase the efficiency of a backcrossing breeding scheme forproducing lettuce line comprising a desired trait. This technique iscommonly referred to as marker assisted backcrossing (MABC). Thistechnique is well-known in the art and may involve, for example, the useof three or more levels of selection, including foreground selection toidentity the presence of a desired locus, which may complement orreplace phenotype screening protocols; recombinant selection to minimizelinkage drag; and background selection to maximize recurrent parentgenome recovery.

D. Plants Derived by Genetic Engineering

Various genetic engineering technologies have been developed and may beused by those of skill in the art to introduce traits in plants. Incertain aspects of the claimed invention, traits are introduced intolettuce plants via altering or introducing a single genetic locus ortransgene into the genome of a recited variety or progenitor thereof.Methods of genetic engineering to modify, delete, or insert genes andpolynucleotides into the genomic DNA of plants are well-known in theart.

In specific embodiments of the invention, improved lettuce lines can becreated through the site-specific modification of a plant genome.Methods of genetic engineering include, for example, utilizingsequence-specific nucleases such as zinc-finger nucleases (see, forexample, U.S. Pat. Appl. Pub. No. 2011-0203012); engineered or nativemeganucleases; TALE-endonucleases (see, for example, U.S. Pat. Nos.8,586,363 and 9,181,535); and RNA-guided endonucleases, such as those ofthe CRISPR/Cas systems (see, for example, U.S. Pat. Nos. 8,697,359 and8,771,945 and U.S. Pat. Appl. Pub. No. 2014-0068797). One embodiment ofthe invention thus relates to utilizing a nuclease or any associatedprotein to carry out genome modification. This nuclease could beprovided heterologously within donor template DNA for templated-genomicediting or in a separate molecule or vector. A recombinant DNA constructmay also comprise a sequence encoding one or more guide RNAs to directthe nuclease to the site within the plant genome to be modified. Furthermethods for altering or introducing a single genetic locus include, forexample, utilizing single-stranded oligonucleotides to introduce basepair modifications in a lettuce plant genome (see, for example Sauer etal., Plant Physiol, 170(4):1917-1928, 2016).

Methods for site-directed alteration or introduction of a single geneticlocus are well-known in the art and include those that utilizesequence-specific nucleases, such as the aforementioned, or complexes ofproteins and guide-RNA that cut genomic DNA to produce a double-strandbreak (DSB) or nick at a genetic locus. As is well-understood in theart, during the process of repairing the DSB or nick introduced by thenuclease enzyme, a donor template, transgene, or expression cassettepolynucleotide may become integrated into the genome at the site of theDSB or nick. The presence of homology arms in the DNA to be integratedmay promote the adoption and targeting of the insertion sequence intothe plant genome during the repair process through homologousrecombination or non-homologous end joining (NHEJ).

In another embodiment of the invention, genetic transformation may beused to insert a selected transgene into a plant of the invention ormay, alternatively, be used for the preparation of transgenes which canbe introduced by backcrossing. Methods for the transformation of plantsare well-known to those of skill in the art and are applicable to manycrop species.

Vectors used for the transformation of lettuce cells are not limited solong as the vector can express an inserted DNA in the cells. Forexample, vectors comprising promoters for constitutive gene expressionin lettuce cells (e.g., cauliflower mosaic virus 35S promoter) andpromoters inducible by exogenous stimuli can be used. Examples ofsuitable vectors include pBI binary vector. The “lettuce cell” intowhich the vector is to be introduced includes various forms of lettucecells, such as cultured cell suspensions, protoplasts, leaf sections,and callus.

A vector can be introduced into lettuce cells by known methods, such asthe polyethylene glycol method, polycation method, electroporation,Agrobacterium-mediated transfer, particle bombardment and direct DNAuptake by protoplasts. See, e.g., Pang et al. (The Plant J., 9, 899-909,1996).

To effect transformation by electroporation, one may employ eitherfriable tissues, such as a suspension culture of cells or embryogeniccallus or alternatively one may transform immature embryos or otherorganized tissue directly. In this technique, one would partiallydegrade the cell walls of the chosen cells by exposing them topectin-degrading enzymes (pectolyases) or mechanically wound tissues ina controlled manner. An example of electroporation of lettuceprotoplasts is presented in Chupeau et al. (Nat. Biotechnol., 7:503-508,1989).

A particularly efficient method for delivering transforming DNA segmentsto plant cells is microprojectile bombardment. In this method, particlesare coated with nucleic acids and delivered into cells by a propellingforce. Exemplary particles include those comprised of tungsten,platinum, and preferably, gold. For the bombardment, cells in suspensionare concentrated on filters or solid culture medium. Alternatively,immature embryos or other target cells may be arranged on solid culturemedium. The cells to be bombarded are positioned at an appropriatedistance below the macroprojectile stopping plate.

An illustrative embodiment of a method for delivering DNA into plantcells by acceleration is the Biolistics Particle Delivery System, whichcan be used to propel particles coated with DNA or cells through ascreen, such as a stainless steel or Nytex screen, onto a surfacecovered with target lettuce cells. The screen disperses the particles sothat they are not delivered to the recipient cells in large aggregates.It is believed that a screen intervening between the projectileapparatus and the cells to be bombarded reduces the size of projectilesaggregate and may contribute to a higher frequency of transformation byreducing the damage inflicted on the recipient cells by projectiles thatare too large.

Microprojectile bombardment techniques are widely applicable, and may beused to transform virtually any plant species. Examples involvingmicroprojectile bombardment transformation with lettuce can be found in,for example, Elliott et al. (Plant Cell Rep., 18:707-714, 2004) andMolinier et al. (Plant Cell Rep., 21:251-256, 2002).

Agrobacterium-mediated transfer is another widely applicable system forintroducing gene loci into plant cells. An advantage of the technique isthat DNA can be introduced into whole plant tissues, thereby bypassingthe need for regeneration of an intact plant from a protoplast. ModernAgrobacterium transformation vectors are capable of replication in E.coli as well as Agrobacterium, allowing for convenient manipulations(Klee et al., Bio-Technology, 3(7):637-642, 1985). Moreover, recenttechnological advances in vectors for Agrobacterium-mediated genetransfer have improved the arrangement of genes and restriction sites inthe vectors to facilitate the construction of vectors capable ofexpressing various polypeptide coding genes. The vectors described haveconvenient multi-linker regions flanked by a promoter and apolyadenylation site for direct expression of inserted polypeptidecoding genes. Additionally, Agrobacterium containing both armed anddisarmed Ti genes can be used for transformation.

In those plant strains where Agrobacterium-mediated transformation isefficient, it is the method of choice because of the facile and definednature of the gene locus transfer. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art (Fraley et al., Nat. Biotechnol., 3:629-635, 1985; U.S.Pat. No. 5,563,055). For example, U.S. Pat. No. 5,349,124 describes amethod of transforming lettuce plant cells using Agrobacterium-mediatedtransformation. By inserting a chimeric gene having a DNA codingsequence encoding for the full-length B.t. toxin protein that expressesa protein toxic toward Lepidopteran larvae, this methodology resulted inlettuce having resistance to such insects.

Transformation of plant protoplasts also can be achieved using methodsbased on calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments (see, e.g.,Potrykus et al., Mol. Gen. Genet., 199:183-188, 1985; Omirulleh et al.,Plant Mol. Biol., 21(3):415-428, 1993; Fromm et al., Nature,312:791-793, 1986; Uchimiya et al., Mol. Gen. Genet., 204:204, 1986;Marcotte et al., Nature, 335:454, 1988). Transformation of plants andexpression of foreign genetic elements is exemplified in Choi et al.(Plant Cell Rep., 13: 344-348, 1994) and Ellul et al. (Theor. Appl.Genet., 107:462-469, 2003).

A number of promoters have utility for plant gene expression for anygene of interest including but not limited to selectable markers,scoreable markers, genes for pest tolerance, disease resistance,nutritional enhancements and any other gene of agronomic interest.Examples of constitutive promoters useful for lettuce plant geneexpression include, but are not limited to, the cauliflower mosaic virus(CaMV) P-35S promoter, which confers constitutive, high-level expressionin most plant tissues (see, e.g., Odel et al., Nature, 313:810, 1985),including monocots (see, e.g., Dekeyser et al., Plant Cell, 2:591, 1990;Terada and Shimamoto, Mol. Gen. Genet., 220:389, 1990); a tandemlyduplicated version of the CaMV 35S promoter, the enhanced 35S promoter(P-e35S) the nopaline synthase promoter (An et al., Plant Physiol.,88:547, 1988), the octopine synthase promoter (Fromm et al., Plant Cell,1:977, 1989); and the figwort mosaic virus (P-FMV) promoter as describedin U.S. Pat. No. 5,378,619 and an enhanced version of the FMV promoter(P-eFMV) where the promoter sequence of P-FMV is duplicated in tandem,the cauliflower mosaic virus 19S promoter, a sugarcane bacilliform viruspromoter, a commelina yellow mottle virus promoter, and other plant DNAvirus promoters known to express in plant cells.

With an inducible promoter the rate of transcription increases inresponse to an inducing agent. Any inducible promoter can be used in theinstant invention. A variety of plant gene promoters that are regulatedin response to environmental, hormonal, chemical, and/or developmentalsignals can be used for expression of an operably linked gene in plantcells, including promoters regulated by (1) heat (Callis et al., PlantPhysiol., 88:965, 1988), (2) light (e.g., pea rbcS-3A promoter,Kuhlemeier et al., Plant Cell, 1:471, 1989; maize rbcS promoter,Schaffner and Sheen, Plant Cell, 3:997, 1991; or chlorophyll a/b-bindingprotein promoter, Simpson et al., EMBO J., 4:2723, 1985), (3) hormones,such as abscisic acid (Marcotte et al., Plant Cell, 1:969, 1989), (4)wounding (e.g., wunl, Siebertz et al., Plant Cell, 1:961, 1989); or (5)chemicals such as methyl jasmonate, salicylic acid, or Safener. It mayalso be advantageous to employ organ-specific promoters (e.g., Roshal etal., EMBO J., 6:1155, 1987; Schernthaner et al., EMBO J., 7:1249, 1988;Bustos et al., Plant Cell, 1:839, 1989). Exemplary organ-specific ororgan-preferred promoters include, but are not limited to, aroot-preferred promoter, such as that from the phaseolin gene(Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. USA, 82:3320-3324,1985); a leaf-specific and light-induced promoter such as that from cabor rubisco (Simpson et al., EMBO J., 4:2723, 1985) and Timko et al.,Nature, 318:579-582, 1985); an anther-specific promoter such as thatfrom LAT52 (Twell et al., Mol. Gen. Genetics, 217:240-245, 1989); apollen-specific promoter such as that from Zm13 (Guerrero et al., Mol.Gen. Genetics, 244:161-168, 1993) or a microspore-preferred promotersuch as that from apg (Twell et al., Sex. Plant Reprod., 6:217-224,1993).

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall, ormitochondrion or for secretion into the apoplast, may be accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized. The presence of asignal sequence directs a polypeptide to either an intracellularorganelle or subcellular compartment or for secretion to the apoplast.Many signal sequences are known in the art. See, for example Becker etal. (Plant Mol. Biol., 20:49, 1992); Knox et al. (Plant Mol. Biol.,9:3-17, 1987); Lerner et al. (Plant Physiol., 91:124-129, 1989); Fonteset al. (Plant Cell, 3:483-496, 1991); Matsuoka et al. (Proc. Natl. Acad.Sci. USA, 88:834, 1991); Gould et al. (J. Cell. Biol., 108:1657, 1989);Creissen et al. (Plant J., 2:129, 1991); Kalderon et al. (Cell,39:499-509, 1984); Steifel et al. (Plant Cell, 2:785-793, 1990).

Exemplary nucleic acids which may be introduced to the lettuce lines ofthis invention include, for example, DNA sequences or genes from anotherspecies, or even genes or sequences which originate with or are presentin the same species, but are incorporated into recipient cells bygenetic engineering methods rather than classical reproduction orbreeding techniques. However, the term “exogenous” is also intended torefer to genes that are not normally present in the cell beingtransformed, or perhaps simply not present in the form, structure, etc.,as found in the transforming DNA segment or gene, or genes which arenormally present and that one desires to express in a manner thatdiffers from the natural expression pattern, e.g., to over-express.Thus, the term “exogenous” gene or DNA is intended to refer to any geneor DNA segment that is introduced into a recipient cell, regardless ofwhether a similar gene may already be present in such a cell. The typeof DNA included in the exogenous DNA can include DNA which is alreadypresent in the plant cell, DNA from another plant, DNA from a differentorganism, or a DNA generated externally, such as a DNA sequencecontaining an antisense message of a gene, or a DNA sequence encoding asynthetic or modified version of a gene.

Many hundreds if not thousands of different genes are known and couldpotentially be introduced into a lettuce plant according to theinvention. Non-limiting examples of particular genes and correspondingphenotypes one may choose to introduce into a lettuce plant include oneor more genes for insect tolerance, such as a Bacillus thuringiensis(B.t.) gene, pest tolerance such as genes for fungal disease control,herbicide tolerance such as genes conferring glyphosate tolerance, andgenes for quality improvements such as yield, nutritional enhancements,environmental or stress tolerances, or any desirable changes in plantphysiology, growth, development, morphology or plant product(s). Forexample, structural genes would include any gene that confers insecttolerance including but not limited to a Bacillus insect control proteingene as described in WO 99/31248, herein incorporated by reference inits entirety, U.S. Pat. No. 5,689,052, herein incorporated by referencein its entirety, U.S. Pat. Nos. 5,500,365 and 5,880,275, hereinincorporated by reference in their entirety. In another embodiment, thestructural gene can confer tolerance to the herbicide glyphosate asconferred by genes including, but not limited to Agrobacterium strainCP4 glyphosate resistant EPSPS gene (aroA:CP4) as described in U.S. Pat.No. 5,633,435, herein incorporated by reference in its entirety, orglyphosate oxidoreductase gene (GOX) as described in U.S. Pat. No.5,463,175, herein incorporated by reference in its entirety.

Alternatively, the DNA coding sequences can affect these phenotypes byencoding a non-translatable RNA molecule that causes the targetedinhibition of expression of an endogenous gene, for example viaantisense- or cosuppression-mediated mechanisms (see, for example, Birdet al., Biotech. Gen. Engin. Rev., 9:207, 1991). The RNA could also be acatalytic RNA molecule (i.e., a ribozyme) engineered to cleave a desiredendogenous mRNA product (see for example, Gibson and Shillito, Mol.Biotech., 7:125,1997). Thus, any gene which produces a protein or mRNAwhich expresses a phenotype or morphology change of interest is usefulfor the practice of the present invention.

E. Definitions

In the description and table herein, a number of terms are used. Inorder to provide a clear and consistent understanding of thespecification and claims, the following definitions are provided:

A: When used in conjunction with the word “comprising” or other openlanguage in the claims, the words “a” and “an” denote “one or more.”

Allele: Any of one or more alternative forms of a genetic locus, all ofwhich alleles relate to one trait or characteristic. In a diploid cellor organism, the two alleles of a given gene occupy corresponding locion a pair of homologous chromosomes.

Backcrossing: A process in which a breeder repeatedly crosses hybridprogeny, for example a first generation hybrid (F₁), back to one of theparents of the hybrid progeny. Backcrossing can be used to introduce oneor more single locus conversions from one genetic background intoanother.

Crossing: The mating of two parent plants.

Cross-pollination: Fertilization by the union of two gametes fromdifferent plants.

Diploid: A cell or organism having two sets of chromosomes.

Emasculate: The removal of plant male sex organs or the inactivation ofthe organs with a cytoplasmic or nuclear genetic factor or a chemicalagent conferring male sterility.

Enzymes: Molecules which can act as catalysts in biological reactions.

F₁ Hybrid: The first generation progeny of the cross of two nonisogenicplants.

Genotype: The genetic constitution of a cell or organism.

Haploid: A cell or organism having one set of the two sets ofchromosomes in a diploid.

Linkage: A phenomenon wherein alleles on the same chromosome tend tosegregate together more often than expected by chance if theirtransmission was independent.

Marker: A readily detectable phenotype, preferably inherited incodominant fashion (both alleles at a locus in a diploid heterozygoteare readily detectable), with no environmental variance component, i.e.,heritability of 1.

Phenotype: The detectable characteristics of a cell or organism, whichcharacteristics are the manifestation of gene expression.

Quantitative Trait Loci (QTL): Quantitative trait loci (QTL) refer togenetic loci that control to some degree numerically representabletraits that are usually continuously distributed.

Regeneration: The development of a plant from tissue culture.

Resistance: As used herein, the terms “resistance” and “tolerance” areused interchangeably to describe plants that show no symptoms to aspecified biotic pest, pathogen, abiotic influence or environmentalcondition. These terms are also used to describe plants showing somesymptoms but that are still able to produce marketable product with anacceptable yield. Some plants that are referred to as resistant ortolerant are only so in the sense that they may still produce a crop,even though the plants are stunted and the yield is reduced.

Royal Horticultural Society (RHS) Colour Chart Value: The RHS ColourChart is a standardized reference which allows accurate identificationof any color. A color's designation on the chart describes its hue,brightness and saturation. A color is precisely named by the RHS ColourChart by identifying the group name, sheet number and letter, e.g.,Yellow-Orange Group 19A or Red Group 41B.

Self-pollination: The transfer of pollen from the anther to the stigmaof the same plant.

Single Locus Converted (Conversion) Plant: Plants which are developed bya plant breeding technique called backcrossing or genetic engineering ofa locus wherein essentially all of the morphological and physiologicalcharacteristics of a lettuce variety are recovered or conserved inaddition to the characteristics of the single locus.

Substantially Equivalent: A characteristic that, when compared, does notshow a statistically significant difference (e.g., p=0.05) from themean.

Tetraploid: A cell or organism having four sets of chromosomes.

Tissue Culture: A composition comprising isolated cells of the same or adifferent type or a collection of such cells organized into parts of aplant.

Transgene: A genetic locus comprising a sequence which has beenintroduced into the genome of a lettuce plant by transformation orsite-specific modification.

Triploid: A cell or organism having three sets of chromosomes.

F. Deposit Information

A deposit of at least 625 seeds of lettuce line SV2867LG, disclosedabove and recited in the claims, has been made with theProvasoli-Guillard National Center for Marine Algae and Microbiota(NCMA), 60 Bigelow Drive, East Boothbay, Me., 04544 USA. The date ofdeposit for those deposited seeds of lettuce line SV2867LG is Sep. 27,2021. The accession number for those deposited seeds of lettuce lineSV2867LG is NCMA Accession Number 202109083. Upon issuance of a patent,all restrictions upon the deposit will be removed, and the deposit isintended to meet all of the requirements of 37 C.F.R. § 1.801-1.809. Thedeposit has been accepted under the Budapest Treaty and will bemaintained in the depository for a period of 30 years, or 5 years afterthe last request, or for the effective life of the patent, whichever islonger, and will be replaced if necessary during that period.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the invention, as limited only bythe scope of the appended claims.

All references cited herein are hereby expressly incorporated herein byreference.

What is claimed:
 1. A lettuce plant of lettuce line SV2867LG, a sampleof seed of said line having been deposited under NCMA Accession Number202109083.
 2. A lettuce seed that produces the plant of claim
 1. 3. Aplant part of the plant of claim 1, wherein the plant part comprises acell of said plant.
 4. A lettuce plant having all the physiological andmorphological characteristics of the plant of claim
 1. 5. A tissueculture of regenerable cells of the plant of claim
 1. 6. A lettuce plantregenerated from the tissue culture of claim 5, wherein said plant hasall of the physiological and morphological characteristics of lettuceline SV2867LG.
 7. A method of vegetatively propagating the plant ofclaim 1, the method comprising the steps of: (a) collecting tissuecapable of being propagated from the plant of claim 1; and (b)propagating a lettuce plant from said tissue.
 8. A method of introducinga trait into a lettuce plant, the method comprising: (a) utilizing as arecurrent parent the plant of claim 1 by crossing the plant with a donorlettuce plant that comprises a trait to produce F1 progeny; (b)selecting an F1 progeny that comprises the trait; (c) backcrossing theselected F1 progeny with a plant of the same lettuce line used as therecurrent parent in step (a) to produce backcross progeny; (d) selectinga backcross progeny comprising the trait and the morphological andphysiological characteristics of the recurrent parent lettuce line usedin step (a); and (e) repeating steps (c) and (d) three or more times toproduce a selected fourth or higher backcross progeny.
 9. A lettuceplant produced by the method of claim
 8. 10. A method of producing alettuce plant comprising an added trait, the method comprisingintroducing a transgene conferring the trait into the plant of claim 1.11. A lettuce plant produced by the method of claim
 10. 12. A lettuceplant of lettuce line SV2867LG, a sample of seed of said line havingbeen deposited under NCMA Accession Number 202109083, further comprisinga transgene.
 13. The plant of claim 12, wherein the transgene confers atrait selected from the group consisting of male sterility, herbicidetolerance, insect resistance, pest resistance, disease resistance,modified fatty acid metabolism, environmental stress tolerance, modifiedcarbohydrate metabolism, and modified protein metabolism.
 14. A lettuceplant of lettuce line SV2867LG, a sample of seed of said line havingbeen deposited under NCMA Accession Number 202109083, further comprisinga single locus conversion.
 15. The plant of claim 14, wherein the singlelocus conversion confers a trait selected from the group consisting ofmale sterility, herbicide tolerance, insect resistance, pest resistance,disease resistance, modified fatty acid metabolism, environmental stresstolerance, modified carbohydrate metabolism, and modified proteinmetabolism.
 16. A method for producing a seed of a lettuce plant derivedfrom lettuce line SV2867LG, the method comprising the steps of: (a)crossing the plant of claim 1 with itself or a second lettuce plant; and(b) allowing a seed of a lettuce line SV2867LG-derived lettuce plant toform.
 17. A method of producing a seed of a lettuce lineSV2867LG-derived lettuce plant, the method comprising the steps of: (a)producing a lettuce line SV2867LG-derived lettuce plant from a seedproduced by crossing the plant of claim 1 with itself or a secondlettuce plant; and (b) crossing the lettuce line SV2867LG-derivedlettuce plant with itself or a different lettuce plant to obtain a seedof a further lettuce line SV2867LG-derived lettuce plant.
 18. The methodof claim 17, the method further comprising repeating said producing andcrossing steps of (a) and (b) using the seed from said step (b) forproducing a plant according to step (a) for at least one generation toproduce a seed of an additional lettuce line SV2867LG-derived lettuceplant.
 19. A method of producing food, the method comprising: (a)obtaining the plant of claim 1, wherein the plant has been cultivated tomaturity, and (b) collecting leaf tissue from the plant, wherein theleaf tissue is capable of use as food.